A number of opiate analgesics currently in use in therapy induce tolerance and physical dependence. Even though a number of in vivo methods are available for monitoring physical dependence potential of opiate analgesics, no reliable in vitro methodology is presently available for screening physical dependence of morphine and its analogs. In the present study we have developed a simple sensitive method for such screening using human neuroblastoma cells (SY-5Y) in culture. Since this cell lines have predominantly mu opiate receptors which has been implicated in morphine induced analgesia and physical dependence, those cells appear to be suitable for this study. In early 1970's Sharma et al have shown that morphine and other analogs of morphine were capable of inhibiting adenylate cyclase in mouse neuroblastoma-glioma hybrid cells (NG108-15) and this inhibition paralleled physical dependence produced by these drugs in vivo. Since NG108-15 cells contain mainly delta opiate receptors we have used SY-5Y cells which have mainly mu opiate receptors. These morphine receptors have been shown to be induced when cells were treated with retionic acid which cause differentiation of these cells. Moreover retinoic acid increased cyclic AMP response to prostaglandins and forskolin - a diterpene which has been shown, to activate adenylate cyclase in cells. Morphine caused a dose dependent inhibition of both PGE1 induced and forskolin activated cyclic AMP formation in SY-5Y cells. The degree of inhibition was higher in forskolin than PGEI induced cyclic AMP formation (75% vs 50% in PGEI induced). Calcium ionophore (A 23187) and ionomycin potentiated forskolin induced cAMP formation and was inhibited by morphine. The effect of morphine on cyclic AMP formation may be mediated by reduction of intracellular calcium. However a direct measurement on calcium level is essential to establish this aspect of morphine. Viminol is a morphine analog which was introduced into therapy some ten years ago. A number of analogs of viminol have been tested in this system. Two of the four analogs show very little inhibition of cyclic AMP formation in contrast to morphine. These new analogs of viminol appear to have very high analgesic activity and may induce little or no physical dependence.
